Selectins are carbohydrate-binding adhesion molecules that, during the process of an immune defense, contribute to an increased adhesion of leukocytes to the vessel endothelium of the inflamed tissue. They are divided into L-(leukocytes), E-(endothelium) and P-(platelets and endothelium) selectin, in accordance with their cells of origin. They initiate the adhesion of leukocytes through their protein structure and their specific molecular binding properties; following a transitional binding of the corresponding ligand, the leukocytes of the flowing blood stream experience a “rolling slowing down” along the wall of the vessel. Subsequently, other adhesion molecules mediate the tight binding of the leukocytes to the endothelium as well as their extravasation, in order to complete their defensive function.
Shortly after their discovery and structural elucidation at the beginning of the 90ties, the selectins became attractive target structures for pharmaceutical research. In addition to their physiological function in the immunological events, also a dysregulation of the expression of selectin during pathological processes, such as rheumatoid arthritis, asthma, diabetes mellitus and ischemia/reperfusion, and a participation in the tissue invasion of metastatic cancer cells was observed. This motivated an intensive search for selectin-inhibiting substances.
Selectin-Ligands
For the selectins as described, only a few high-affinity ligands are known. In principle, these represent mucin-like structures, that is, long-stretched glycoproteins, to which many carbohydrate-side chains are glycosidically bound to the serine- or threonine-rich protein matrix thereof as the actual binding epitopes. Through a rapid formation and dissociation of the receptor binding to the highly flexible ligand, the rolling of the cells is mediated in the shearing flow of the vessels. The carbohydrate-epitopes that are essential for the binding are N-acetyllactosamine-based oligosaccharides in specific linkage with fucose and a terminal sialic acid (N-acetyl-neuraminic acid). The tetrasaccharide sialyl Lewis X (sLeX) is of particular importance as a binding epitope.
Although all selectins bind sLeX with only very low affinity (about 1 mM), yet, no carbohydrates having an improved binding have been found, therefore, the high binding affinity to the natural mucin ligands (about 100 nM) can not be structurally explained. Against this background, sLeX was used as a standard ligand for structure-effect-relationships, in order to characterize the binding properties as well as for the search for selectin-inhibitors.
Modification of the Lead Structure Sialyl Lewis X
Due to the fact that the tetrasaccharide sialyl Lewis X (sLeX) is able to bind to all three selecting, it functions as central lead substance for the search for selectin inhibiting compounds. Based on the x-ray structural data of the E-selectin (Graves, B. J., et al., Insight into E-selectin/ligand interaction from the crystal structure and mutagenesis of the lec/EGF domains. Nature 367 (1994) 532-538.), and several NMR-examinations of the dissolved sLeX, or sLeX bound to E-selectin (Cooke, R. M., et al. The conformation of the sialyl Lewis x ligand changes upon binding to E-Selectin. Biochemistry 33 (1994) 10591-10596; Kogan, T. P., et al., A single amino acid residue can determine the ligand specificity of E-Selectin. J. Biol. Chem. 270 (1995) 14047-14055.), the structural elements that are essential for the binding of the lectin domain could be identified: the negative charge of the sialic acid, the 2-, 3-, and 4-positioned hydroxy group of the fucose, as well as the 6-positioned hydroxy group of the galactose. Introducing structural modifications while maintaining the structural features as just mentioned, it was tried to obtain selectin inhibitors that exhibit an improved binding. The disadvantages of glycosidic inhibitors are the complex synthesis pathway, the high costs of the starting compounds, the difficult structural elucidation, the hydrolytic instability, and the relatively low binding affinity. For an extensive review of synthetic selectin inhibitors, reference shall be made to Simanek et al. (Simanek, E. E., et al., Selectin-carbohydrate interactions: From natural ligands to designed mimetics. Chem. Rev. 98 (1998) 833-862.).
The simplest synthetic modification of the sLeX consists in a replacement of the negatively charged sialic acid through simpler negatively charged structural elements. Thus, sulfur-, phosphor-, and carboxylic acid-derivatives at the C3 of the galactose have been described. These structural variations resulted in similar binding affinities such as the one for sLeX itself (Ohmoto, H., et al., Studies on selectin blockers 1: Structure activity relationships of Sialyl Lewis x analogs. J. Med. Chem. 39 (1996) 1339-1343.).
A further simplification consists in the substitution of the N-acetylated glucose that does not contribute to the binding, but is merely responsible for the optimal orientation of the overall structure. The substitution of the glucose by alkanedioles or cyclohexanedioles without or with simultaneous substitution of the sialic acid led to derivatives that, in some cases, also exceeded the effectivity of sLeX (Norman, K. E., et al., Sialyl Lewis X (sLex) and an sLex mimetic, CGP69669A, disrupt E-selectin-dependent leukocyte rolling in vivo. Blood 91 (1998) 475-483.). The simultaneous substitution of glucose and galactose gave rigid and stabile fucose-sialic acid-derivatives, which, due to the lack of the galactose-6-hydroxy group, nevertheless, gave no improved effect.
The largest group by number of the sLeX-analogs as tested consists of monosaccharide-compounds, wherein, while maintaining the fucose, the other essential binding epitopes in the molecule were replaced by non-glycosidic structures. Most successful are molecules, wherein specific peptides at the fucose simulate the contribution to the binding of the galactose and the negative sialic acid. Some substances exhibit a similar, in individual cases even a markedly enhanced binding affinity to the selectins, compared with the sLeX (Wong, C. H., et al., Small molecules as structural and functional mimics of sLex tetrasaccharide in selectin inhibition: A remarkable enhancement of inhibition by additional negative charge and for hydrophobic groups. J. Am. Chem. Soc. 119 (1997) 8152-8158.). Starting from sLeX, additional binding epitopes were searched for. It was found through the derivatization of the sLeX, and the computer simulation of the binding at the lectin domain, that a hydrophobic substitution at the glycosidic hydroxy group or at the amine of the glucosamine drastically improved the binding properties. New derivatives with aromatic ring systems at the amine or branched chain fatty acids at the glycosidic residue of the glucosamines, but also simplified sLeX-analogs with fatty acid substitutions were successfully tested (Ramphal, J. Y., et al., Ligand interactions with E-Selectin. Identification of a new site for recognition of N-Acyl Aromatic Glucosamine substituents of Sialyl Lewis x. J. Med. Chem. 39 (1996) 1357-1360.), and partially exhibited a more than 10-fold stronger binding, when compared with sLeX.
Another theory related to the enhancement of the binding is based on the joint cooperation of several singular bonds. This multi-valence hypothesis is also discussed as the underlying and effective principle of the natural selectin ligands. Based on this, several oligo- and polymeric derivatives were synthesized, wherein sLeX-molecules (Renkonen, O., et al., Synthesis of new nanomolar saccharide inhibitors of lymphocyte adhesion: Different polylactosamine backbones present multiple Sialyl Lewis X determinants to L-Selectin in high affinity mode. Glycobiology 7 (1997) 453-461.), or their structurally simplified glycosides (Manning, D. D., et al., Neoglycopolymer inhibitors of the selecting. Tetrahedron 53 (1997) 11937-11952.), are sterically tightly connected through carrier molecules. Despite the partially improved binding parameters, the multi-valence hypothesis is differently discussed. Binding studies using a multimeric, non-covalently fixed sLeX-array in liposomes (Stahn, R., et al., Multivalent sLex ligands of definite structure as inhibitors of E-selectin mediated cell adhesion. Glycobiology 8 (1998) 311-319) or in planar model membranes (Vogel, J., et al., The role of glycolipids in mediating cell adhesion: A flow chamber study. Biochim. Biophys. Acta 1372 (1998) 205-215.), support the hypothesis.
Aggravating for a valuing comparison of all selectin inhibitors as synthesized is the fact that different test systems are used in the individual research groups.
Until now, only one selectin inhibiting substance having a glycosidic structure can be found in clinical trials (phase IIa), namely the pan-selectin antagonist bimosiamose (1,6-bis[3-(3-carboxymethylphenyl)-4-(2-alpha-D-mannopyranosyloxy)phenyl]hexane), which exhibits an essentially higher affinity to the selectins compared to sLeX. The Revotar AG is testing bimosiamose for the treatment of psoriasis and atopic dermatitis (Ulbrich H, et al. Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inflammatory disease. Trends Pharmacol Sci. (2003) 640-647.).
It is therefore an object of the present invention to provide inhibitors of selectin which overcome the disadvantages of inhibitors of selectin that are known in the state of the art, and which, in addition are more stable and synthetically more accessible.
According to the invention, this object is solved by providing compounds of the general formula 1
wherein    n is 0 or 1,    R1 to R6 independently are H, COOH, COOCH3, COOC2H5 or halogen, and    X is C═N—O—(CH2)m—Y or N—C(═O)—(CH2)m—Y,wherein    m is 5 or 6, and    Y is
wherein    R7 is H or O(CH2)9CH3,or derivatives thereof, their diastereomers, and pharmaceutically acceptable salts of said compounds.
Thereby, preferred are compounds, that are derived from formula 2, wherein formula 2 is
wherein    R1 is H,    R2 is H, COOH, COOCH3 or halogen,    R3 is H, COOH or COOCH3,    R4 is H,    R5 is H, COOH, COOCH3 or halogen,    R6 is H, and    R7 is H or O(CH2)9CH3,or derivatives thereof, their diastereomers, and pharmaceutically acceptable salts of said compounds.
Furthermore preferred are compounds, that are derived from formula 3, wherein formula 3 is
wherein    n is 0 or 1,    R1 is H, COOH or COOCH3,    R2 is H, COOH, COOCH3 or COOC2H5,    R3 is H, COOH or COOCH3,    R4 is H, COOH or COOCH3,    R5 is H, COOH, COOCH3 or COOC2H5,    R6 is H, and    R7 is H or O(CH2)9CH3,or derivatives thereof, their diastereomers, and pharmaceutically acceptable salts of these compounds.
A preferred embodiment of the present invention is represented by pharmaceutical compositions that comprise at least one compound according to the formulae 1-3, and suitable additives or auxiliary agents. Such suitable additives and auxiliary agents are known to the person of skill and from the state of the art.
In these pharmaceutical compositions, the compounds can be present in form of a depot substance or as a precursor, together with a suitable, pharmaceutically acceptable diluent or carrier substance.
It is preferred that the compounds are present in a pharmaceutical composition in an amount of 0.1 to 1000 mg, more preferred of 1 to 100 mg per dosage unit.
Furthermore, additional selectin inhibitors can be contained in the pharmaceutical compositions. These additional selectin inhibitors can comprise usual selectin inhibitors that are known to the person of skill, such as, for example, sialyl Lewis X (sLeX), a large number of sLeX-analogs, such as, for example, monosaccharide and oligosaccharide-compounds, and glycosidic selectin inhibitors, such as, for example, bimosiamose. Synthetic selectin inhibitors can be used (for this, see also: Simanek, E. E., et al., Selectin-carbohydrate interactions: From natural ligands to designed mimetics. Chem. Rev. 98 (1998) 833-862.).
Preferred forms of the pharmaceutical compositions are tablets, dragees, capsules, droplets, suppositories, preparations for injection or infusion for peroral, rectal or parenteral use. Such administration forms and their production are known to the person of skill.
It is preferred that the compounds are present in a pharmaceutical composition in such an amount that an amount in a concentration range of between 0.1 and 100 μM can be found during a treatment in vivo.
The method according to the invention for the synthesis of a compound according to the formulae 1 to 3 preferably comprises the synthesis from 1-(6-bromohexyloxy)-3,5-didecyloxybenzene and a compound selected from the group of bis(4-methoxycarbonylphenyl)methanone, N N-bis(4-methoxycarbonylphenyl)amine, and N-(4-carboxybenzyl)-N-(4-carboxyphenyl)amine.
In doing so, it is preferred that a compound according to formula 2 is synthesized from bis(4-methoxycarbonylphenyl)methanone (compound 6 in scheme 1) and 1-(6-bromohexyloxy)-3,5-didecyloxybenzene (compound 10 in scheme 1).
It is furthermore preferred that the production of a compound according to formula 3 comprises the synthesis from N,N-bis(4-methoxycarbonylphenyl)amine (compound 15 a in scheme 2) or N-(4-carboxybenzyl)-N-(4-carboxyphenyl)amine (compound 15 b in scheme 2) and 1-(6-bromohexyloxy)-3,5-didecyloxybenzene (compound 10 in scheme 1).
Preferably, a synthesis of the compounds according to the invention takes place as illustrated in the schemes 1 and 2.
The present invention provides a novel class of non-glycosidic and non-peptidic selectin inhibitors with low molecular weight. These were synthesized as ligands for an inhibition of the selectin-mediated leukocyte adhesion in accordance with different strategies (schemes 1 + 2), based on a pharmacophoric model including molecular modeling methods.

aReagents and reaction conditions: (a) i-PrMgCl, THF, 40 min, −30° C. to −20° C.; (b) 4-formylbenzoic acid methylester (16), THF, −20° C.; RT, over night; (c) PCC, CH2Cl2, 24 h, RT; (d) HCl.H2N—OH, EtOH, NaOAc, 24 h, reflux; (e) 1-decanol, gaseous HCl, 3 h, 70° C.; (f) 1,6-dibromohexane, K2CO3, cyclohexanone, 24 h, reflux; (g) NaH, DMF; 10, n-BU4NI, 2 h, RT; (h) 0.5 M NaOH, EtOH/THF, 24 h, RT; (see example 8).

aReagents and reaction conditions: (a) Cu, CuI, K2CO3, n-Bu2O, 4 d, reflux; (b) NaOCH3, CH3OH, 1.5 h, reflux; (c) MeOH, 1 h, reflux; (d) NaBH4, MeOH, 24 h, RT; (e) NaH, n-Bu2O, 2 h, RT; 6-bromohexanoic acid chloride, 4 h, reflux; (f) 9, K2CO3, KI, cyclohexanone, 5 h, reflux; (g) 0.5 M LiOH, THF, 8 h, 5° C., then 16 h, 15° C.; (see example 8).
Subsequently, the compounds as produced according to the invention can be derivatized. Preferred derivatizations comprise the formation of ester-, amide-, and hydroxamic acid-derivatives of the compounds according to the invention.
Preferred members of the compounds that can be derived from the formulae 1-3 comprise the compounds that are shown in the overviews 1 and 2 for the formulae.
Overview 1 for formulaeformula (2)T2 (syn.: 11)R1 = HR2 = COOCH3R3 = HR4 = HR5 = COOCH3R6 = HR7 = O(CH2)9CH3T4 (syn.: 1)R1 = HR2 = COOHR3 = HR4 = HR5 = COOHR6 = HR7 = O(CH2)9CH3T8R1 = HR2 = HR3 = COOHR4 = HR5 = COOHR6 = HR7 = O(CH2)9CH3T9R1 = HR2 = HR3 = COOCH3R4 = HR5 = COOCH3R6 = HR7 = O(CH2)9CH3T10R1 = HR2 = ClR3 = HR4 = HR5 = ClR6 = HR7 = O(CH2)9CH3T27R1 = HR2 = COOHR3 = HR4 = HR5 = COOHR6 = HR7 = H
Overview 2 for formulaeformula (3)T1n = 0R1 = HR2 = COOC2H5R3 = HR4 = HR5 = COOC2H5R6 = HR7 = O(CH2)9CH3T3 (syn.: 2a)n = 0R1 = HR2 = COOHR3 = HR4 = HR5 = COOHR6 = HR7 = O(CH2)9CH3T5 (syn.: 20a)n = 0R1 = HR2 = COOCH3R3 = HR4 = HR5 = COOCH3R6 = HR7 = O(CH2)9CH3T6n = 0R1 = HR2 = HR3 = COOCH3R4 = HR5 = COOCH3R6 = HR7 = O(CH2)9CH3T7n = 0R1 = HR2 = HR3 = COOHR4 = HR5 = COOHR6 = HR7 = O(CH2)9CH3T17 (syn.: 2b)n = 1R1 = HR2 = COOHR3 = HR4 = HR5 = COOHR6 = HR7 = O(CH2)9CH3T18 (syn.: 20b)n = 1R1 = HR2 = COOCH3R3 = HR4 = HR5 = COOCH3R6 = HR7 = O(CH2)9CH3T20n = 1R1 = COOHR2 = HR3 = HR4 = HR5 = COOHR6 = HR7 = O(CH2)9CH3T22n = 1R1 = HR2 = COOCH3R3 = HR4 = HR5 = COOCH3R6 = HR7 = HT23n = 0R1 = HR2 = COOHR3 = HR4 = COOHR5 = COOHR6 = HR7 = O(CH2)9CH3T24n = 1R1 = COOCH3R2 = HR3 = HR4 = HR5 = COOCH3R6 = HR7 = HT25n = 0R1 = HR2 = COOCH3R3 = HR4 = COOCH3R5 = COOCH3R6 = HR7 = O(CH2)9CH3T26n = 1R1 = COOHR2 = HR3 = HR4 = HR5 = COOHR6 = HR7 = HT28n = 1R1 = HR2 = COOHR3 = HR4 = HR5 = COOHR6 = HR7 = HT21:4-(N-(6-(3,5-bis(decyloxy)phenoxy)hexyl)-N-(4-carboxyl-phenyl)amino)benzoic acid; C46H67NO7 (T21)
Said 4-[(N-4-carboxyphenyl-N-acyl)amino]benzoic acid-derivatives or 4-[(N-4-carboxyphenyl-N-acyl)imino]benzoic acid-derivatives, respectively, as mimetics of sLeX, have an essentially higher affinity (f ≈6 in the static assay, see example 1) for the selecting, compared to the above-mentioned bimosiamose itself. These mimetics contain functional groups, such as, for example, hydroxy groups and acidic units, in order to mimic fucose and sialic acid, aromatic regions as substitutions for lactosamine, having a defined distance from lipophilic and hydrophilic structural elements.
The results of a static leukocyte-adhesion-assay using compounds T1 to T27 according to the invention (see FIG. 1 as well as example 1) clearly show that the most potent of the compounds according to the invention are carboxylic acids. Thus, different carboxylic acids, each having an amide-, oxime-, and amino-structural element, respectively, were produced and tested. With the exception of T25, all esters only exhibited a weak effect on the reduction of the adhesion. As a comparison: bimosiamose, in a concentration of 600 μM, has about the same potency as T3 in a concentration von 100 μM.
In addition, in a mouse-peritonitis-model, a model of acute inflammation, it could be exemplary shown that the substances according to the invention led to a reduction of the infiltration of polymorphonuclear leukocytes (PMNs) into the peritoneum (see FIG. 2 and example 7), and thus are anti-inflammatory effective.
Furthermore, in toxicity-assays those structural elements were already identified that could be responsible for a cytotoxicity of the compounds according to the invention (see FIG. 3 and example 6).
The compounds according to the invention that can be derived from the formulae 1 to 3 can be used as medicaments.
In doing so, preferred is the use for the treatment of diseases, wherein the selectin-mediated leukocyte adhesion is dysregulated.
Furthermore preferred is the use for the treatment of inflammatory diseases. The inflammatory diseases that can be treated according to the invention comprise peritonitis and rheumatoid arthritis. Furthermore, the treatment of tumorous diseases is possible.
The compounds according to the invention are preferably used as inhibitors of selectin and for an inhibition of the selectin-mediated leukocyte adhesion.
In summary, it can be said that the present invention comprises a novel class of non-toxic, in vivo anti-inflammatory effective, non-glycosidic and non-peptidic inhibitors of selectin that do not exhibit the disadvantages of the glycosidic inhibitor complexes, such as 1. the complex synthesis pathway, 2. the high costs of the starting compounds 3. the laborious elucidation of the structure, 4. the hydrolytic instability, and 5. the relatively low binding affinity, and that are furthermore more potent in vitro than the drug bimosiamose which is currently in the clinical phase II.
In the context of the present invention, a “derivative” shall mean a compound, which is derived from one of the general formulae 1, 2 or 3 that, for example, is substituted with several of the residual groups as given above for R1 to R6 and X or Y, as well as mixtures of several of these compounds, which, for example, can be processed into a “personalized” medicament which is adjusted to with respect to the respective disease to be treated and/or the patient on the basis of diagnostic data, or data with respect to the success or progression of the treatment. A derivative shall also include a compound that can be produced in accordance with a synthesis pathway that is different from the one that is (exemplary) mentioned herein. Preferred derivatizations comprise the formation of ester-, amide- and hydroxamic acid-derivatives of the compounds according to the invention.
In the context of the present invention, a “precursor” of a substance shall first mean a substance that, during the administration for the treatment, is modified by the conditions inside the body (e.g. pH in the stomach, or the like) in such a manner, or, after uptake into the body, is metabolized in such a manner that the compounds according to the invention or their derivatives are formed as effective substances.